2A self-cleaving peptides

2A self-cleaving peptides, or 2A peptides, is a class of 18–22 aa-long peptides, which can induce the cleaving of the recombinant protein in a cell.[1][2] These peptides share a core sequence motif of DxExNPGP, and are found in a wide range of viral families. They help break apart polyproteins by causing the ribosome to fail at making a peptide bond.[3][4]

An illustration of 2A peptide function: when the CDS of a 2A peptide is inserted between two CDSs of protein, the peptide will be cleaved into two proteins folding independently.

The members of 2A peptides are named after the virus in which they have been first described. For example, F2A, the first described 2A peptide, is derived from foot-and-mouth disease virus. The name "2A" itself comes from the gene numbering scheme of this virus.[1][5]

Members

Four members of 2A peptides family are frequently used in life science research. They are P2A, E2A, F2A and T2A. F2A is derived from foot-and-mouth disease virus 18; E2A is derived from equine rhinitis A virus; P2A is derived from porcine teschovirus-1 2A; T2A is derived from thosea asigna virus 2A.[1]

The following table shows the sequences of four members of 2A peptides. Adding the optional linker “GSG” (Gly-Ser-Gly) on the N-terminal of a 2A peptide helps with efficiency.[6]

NameSequence
T2A(GSG) EGRGSLL TCGDVEENPGP
P2A(GSG) ATNFSLLKQAGDVEENPGP
E2A(GSG) QCTNYALLKLAGDVESNPGP
F2A(GSG) VKQTLNFDLLKLAGDVESNPGP

Description

The cleavage is trigged by breaking of peptide bond between the Proline (P) and Glycine (G) in C-terminal of 2A peptide. The exact molecular mechanism of 2A-peptide-mediated cleavage is still unknown.[7][8] However, it is believed to involve ribosomal "skipping" of glycyl-prolyl peptide bond formation rather than true proteolytic cleavage.[9][10]

Application

In genetic engineering, the 2A peptides are used to cleave a longer peptide into two shorter peptides. The 2A peptides can be applied when the fused protein doesn’t work. Inserting the CDS of a 2A peptide into the fusing point or replacing the linker sequence with the CDS of a 2A peptide protein can cleave the fused protein into two separated peptides, making the two peptides to regain the function.[6]

2A peptides, when combined with the IRES elements, can make it possible to generate four separated peptides within a single transcript.[1]

Incomplete cleavage

Different 2A peptides have different efficiencies of self-cleaving, P2A being the most and F2A the least efficient.[11] Therefore, up to 50% of F2A-linked proteins can remain in the cell as a fusion protein, which might cause some unpredictable outcomes, including a gain of function.[12]

See also

References
  1. Liu, Ziqing; Chen, Olivia; Wall, J. Blake Joseph; Zheng, Michael; Zhou, Yang; Wang, Li; Ruth Vaseghi, Haley; Qian, Li; Liu, Jiandong (2017). "Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector". Scientific Reports. 7 (1): 2193. Bibcode:2017NatSR...7.2193L. doi:10.1038/s41598-017-02460-2. ISSN 2045-2322. PMC 5438344. PMID 28526819.
  2. Sampath Karuna; Roy Sudipto (30 August 2010). Live Imaging In Zebrafish: Insights Into Development And Disease. World Scientific. pp. 51–52. ISBN 978-981-4464-89-5.
  3. Luke, Garry A.; de Felipe, Pablo; Lukashev, Alexander; Kallioinen, Susanna E.; Bruno, Elizabeth A.; Ryan, Martin D. (1 April 2008). "Occurrence, function and evolutionary origins of '2A-like' sequences in virus genomes". Journal of General Virology. 89 (4): 1036–1042. doi:10.1099/vir.0.83428-0. PMC 2885027. PMID 18343847.
  4. Yang, X; Cheng, A; Wang, M; Jia, R; Sun, K; Pan, K; Yang, Q; Wu, Y; Zhu, D; Chen, S; Liu, M; Zhao, XX; Chen, X (2017). "Structures and Corresponding Functions of Five Types of Picornaviral 2A Proteins". Frontiers in Microbiology. 8: 1373. doi:10.3389/fmicb.2017.01373. PMC 5519566. PMID 28785248.
  5. Ryan, MD; King, AM; Thomas, GP (November 1991). "Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence". The Journal of General Virology. 72 ( Pt 11) (11): 2727–32. doi:10.1099/0022-1317-72-11-2727. PMID 1658199.
  6. Szymczak-Workman, A. L.; Vignali, K. M.; Vignali, D. A. A. (2012). "Design and Construction of 2A Peptide-Linked Multicistronic Vectors". Cold Spring Harbor Protocols. 2012 (2): 199–204. doi:10.1101/pdb.ip067876. ISSN 1559-6095. PMID 22301656.
  7. Wang, Yuancheng; Wang, Feng; Wang, Riyuan; Zhao, Ping; Xia, Qingyou (2015). "2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori". Scientific Reports. 5 (1): 16273. Bibcode:2015NatSR...516273W. doi:10.1038/srep16273. ISSN 2045-2322. PMC 4633692. PMID 26537835.
  8. "Cleavage Activity of Aphtho- and Cardiovirus 2A Oligopeptidic Sequences". University of St Andrews. Archived from the original on 2016-12-30. Retrieved 2019-01-05.
  9. Ryan, Martin D.; Mehrotra, Amit; Gani, David; Donnelly, Michelle L. L.; Hughes, Lorraine E.; Luke, Garry; Li, Xuejun (1 May 2001). "Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'". Journal of General Virology. 82 (5): 1013–1025. doi:10.1099/0022-1317-82-5-1013. PMID 11297676.
  10. Sharma, Pamila; Yan, Fu; Doronina, Victoria A.; Escuin-Ordinas, Helena; Ryan, Martin D.; Brown, Jeremy D. (April 2012). "2A peptides provide distinct solutions to driving stop-carry on translational recoding". Nucleic Acids Research. 40 (7): 3143–3151. doi:10.1093/nar/gkr1176. PMC 3326317. PMID 22140113.
  11. Kim, Jin Hee; Lee, Sang-Rok; Li, Li-Hua; Park, Hye-Jeong; Park, Jeong-Hoh; Lee, Kwang Youl; Kim, Myeong-Kyu; Shin, Boo Ahn; Choi, Seok-Yong (2011-04-29). Thiel, Volker (ed.). "High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice". PLOS ONE. 6 (4): e18556. Bibcode:2011PLoSO...618556K. doi:10.1371/journal.pone.0018556. ISSN 1932-6203. PMC 3084703. PMID 21602908.
  12. Velychko, Sergiy; Kang, Kyuree; Kim, Sung Min; Kwak, Tae Hwan; Kim, Kee-Pyo; Park, Chanhyeok; Hong, Kwonho; Chung, ChiHye; Hyun, Jung Keun (April 2019). "Fusion of Reprogramming Factors Alters the Trajectory of Somatic Lineage Conversion". Cell Reports. 27 (1): 30–39.e4. doi:10.1016/j.celrep.2019.03.023. PMID 30943410.
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